Enzyme hydrolysates of cow's milk or fractions of cow's milk have only limited application in the food industry. Nevertheless, these hydrolysates occupy interesting niches in the marketplace, as evidenced by the large volume of literature describing and claiming optimised processes for obtaining such hydrolysates. Milk or milk fractions are subjected to enzymes having proteolytic activity to produce the hydrolysates primarily to minimize the allergenicity of the product, facilitate gastrointestinal uptake by offering an easily assimilable digest, and to stabilize the proteins in acid products against precipitation during prolonged storage periods.
Although reducing the molecular weight of milk proteins is commonly accepted practice for producing these beneficial effects, enzymatic hydrolysis of milk proteins does have drawbacks. Negative aspects of incubating milk with enzymes include incomplete proteolytic digestion, an increasingly bitter taste upon decreasing the length of the peptide fragments, decreased yields of the final product due to the requisite purification steps, and unpleasant taste changes caused by high levels of free amino acids.
Uniform and complete degradation of all milk fractions via incubation with endoproteases is often difficult to obtain. For example, beta-lactoglobulin is known to be protease resistant and partial digests of this molecule can lead to unexpectedly strong immunogenic reactions to infant formulae, as well as visible protein precipitations in products such as acidic sport drinks. To guarantee the absence of inadequately digested proteins in a protein hydrolysate, a final ultrafiltration step for the removal of any remaining large peptide fragments from the hydrolysate is generally required. The indispensable step of removing these partially digested protein fragments from the hydrolysate inevitably lowers the yield of the final digestion product, thereby increasing production costs.
Protein antigenicity may be overcome by digesting proteins to peptides having only 8-10 amino acid residues, but the peptides created by such an extensive proteolytic digestion can be very bitter. The general explanation for this phenomenon is that smaller peptides with a high content of hydrophobic amino acids promote bitter tastes. The nature of the proteinaceous raw material used, the type of proteolytic enzymes used for digestion and the length of peptides obtained largely determine the degree of bitterness associated with the final hydrolysate. For example, casein, which contains many hydrophobic amino acids, is known to generate far more bitter hydrolysates than whey proteins.
In industrial operations, debittering of protein hydrolysates is carried out by the selective removal of bitter peptides using activated carbon or adsorption to hydrophobic resin. The concomitant yield reduction during such removal steps increases the cost of the final product. Moreover, this process has a negative impact on the nutritional value of the final product, as several nutritonally indispensable amino acids may be lost due to their hydrophobic nature, including tryptophan, leucine, phenylalanine and isoleucine. Thus, debittering in this way is prone to producing hydrolysates deficient in these nutritionally important amino acids.
Debittering can also be achieved by subjecting hydrolysates to exopeptidases. In this approach, amino-terminal and carboxy-terminal amino acids are liberated from peptides in an attempt to reduce their overall hydrophobicity. Exposure of peptides to non-selective exoproteases unfortunately results in the release of uncontrollable quantities of free amino acids into the final hydrolysate. Subsequent heating of such hydrolysates containing free amino acids, as required for sterilisation or spray drying, often generates brothy off-flavours via Maillard reactions. Moreover, the high levels of free amino acids created by exoproteases may increase the osmotic value of the final hydrolysate product to levels that can cause osmotic diarrhoea.
Therefore, the production of protein hydrolysates represents a trade-off between the pros and cons of proteolytic digestion. Current practise is to optimize enzymatic digestion of protein substrates for the particular requirements of a product category. For example, protein hydrolysates intended for truly allergic infants require extensive proteolytic digestion followed by a rigorous removal of any remaining large molecular weight peptide fragments. By contrast, products designed for adults, who rarely exhibit bovine milk allergies, typically contain hydrolysates in which the average peptide length is increased to minimize the possibility of off flavours and to maximize product yield.
All major milk proteins, such as beta-casein, beta-lactoglobulin and alpha-lactoalbumin, as well as vegetable protein fractions obtained from, for example, soy isolates, rice proteins and wheat gluten are considered important antigenic compounds. Thus, enzymatic digestion of these milk and cereal proteins to molecular weights below 3000 Da is considered important to minimize allergenicity. The beta-lactoglobulin fraction in whey is especially thought to be an important allergen because this protein is not present in human milk and proteolytic digestion of beta-lactoglobulin has proven to be difficult. Infant formula containing protein hydrolysates that are extensively hydrolyzed typically contain high levels of free amino acids, which are indicative of suboptimal taste and high osmolalities. Recent evaluations of currently marketed hydrolyzed infant formula products have shown that most of them still contain whey based immunogenic materials. This observation indicates that new enzyme mixtures leading to improved hydrolysates at a lower cost continue to be in demand.
Protein hydrolysates in products destined for consumers with non-medical needs, for example athletes or people on a slimming diet, must be tailored to provide good taste characteristics. Under these circumstances, high palatability as well as physico-chemical aspects, such as solubility under acidic conditions, are of overriding importance. Products in this category, including fortified fruit juices and sports drinks, focus on, inter alia, glutamine and arginine supplementation to improve consumer health. Sports drinks, for example, serve to enhance physical endurance and recovery of an athlete after prolonged high intensity exercise. Glutamine-rich cereal protein sources, like wheat gluten, or arginine-rich protein sources, like rice protein and soy isolates, have been considered as alternatives to milk proteins to satisfy the supplementation needs of acidic health-related products. However, such cereal proteins, particularly wheat gluten, exhibit very poor solubilities at more acidic pH values i.e. those above 4, meaning completely soluble gluten hydrolysates are difficult to obtain.
Because of the negative influence on product cost and quality associated with protein hydrolysis, several enzyme mixtures aimed at improving hydrolysate characteristics and lowering production costs have been described in prior publications. Examples include EP 321 603, which refers to the use of animal-derived endoproteases like trypsin, chymotrypsin and pancreatin, and EP 325 986 and WO 96/13174, which favor the use of endoproteases obtained from Bacillus or Aspergillus species. Several exoproteases have been described as being capable of debittering mixtures of peptides. Whereas, for example, EP 0223 560 refers to the use of a specific proline specific endoprotease, WO 96/13174 refers to a mixture of amino-peptidases and carboxypeptidases for this purpose.
A number of publications tout the beneficial effects of proline-specific endoproteases in combination with various exopeptidases for producing protein hydrolysates which have relatively low bitterness profiles. For example, Japanese patent JP02039896 refers to the use of a proline-specific endoprotease combined with a dipeptidyl-carboxypeptidase for generating low molecular weight peptide preparations. The degradation of proline-rich oligopeptides by three proline-specific peptide hydrolases is described as essential for accelerating cheese ripening without bitterness (Journal of Dairy Science, 77 (2) 385-392 (1994)). More specifically, the debittering effect of proline-specific endoprotease in combination with a carboxypeptidase is described in JP5015314. JP5015314 describes a crude enzyme preparation obtained from Aspergillus oryzae that exhibits, apart from a general, non-specific proteolytic activity, small quantities of a proline-specific endoprotease and carboxypeptidase activity. According to JP5015314, proline residues present at the carboxy terminii of peptides cause bitter tastes and are undesirable. Incubation of soy bean protein with a proline-specific endoprotease and carboxypeptidase enzyme mixture yielded a hydrolysate that was significantly less bitter than a soy bean hydolysate obtained with protease preparation lacking the combination of a proline-specific endoprotease and a carboxypeptidase.
Collectively, the state of the art strongly suggests that exopeptidase-mediated release of carboxy terminal (or amino terminal) hydrophobic amino acid residues from peptides is essential for significantly debittering peptide hydrolysates. Likewise, references that specifically refer to proline-specific endoproteases for debittering teach that the function of this activity is to expose the hydrophobic proline residues to allow their subsequent removal by a carboxypeptidase. The implication of this hypothesis is that the debittering activity of proline-specific endoproteases is linked with the efficient removal of the carboxy terminal proline residues rather than the creation of peptides carrying such carboxy terminal proline residues.